A major challenge facing biological researchers in the 21st century is the functional characterization of the large number of unannotated gene products identified by genome sequencing efforts. Many proteins partly or completely uncharacterized with respect to their biochemical activities belong to expansive, sequence-related families. Although such membership can inform on the general mechanistic class to which a protein belongs (e.g., enzyme, receptor, channel), it is insufficient to predict specific biochemical and physiological functions, which requires knowledge of substrates, ligands, and interacting biomolecules. On the contrary, membership within a large protein family can even present a barrier to achieving these goals by frustrating the implementation of standard genetic and pharmacological methods to probe protein function. For example, targeted gene disruption of one member of a protein superfamily may result in cellular compensation from other family members.
Problems are also encountered when attempting to develop specific inhibitors and/or ligands for uncharacterized members of large protein families, where at least two major experimental issues must be addressed. First, there is an intrinsic difficulty facing ligand discovery for uncharacterized proteins, which often lack the functional information required to develop high quality assays for compound screening. Second, even with a general screening assay in hand, achieving ligand selectivity for one member of a large protein family presents a major challenge.
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, due in large part to a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems.
As disclosed in U.S. Provisional Application Ser. No. 61/407,732, filed Oct. 28, 2010, which is incorporated herein by reference in its entirety, certain of the inventors herein have developed library versus library screening techniques based on activity-based probes that allow identification of candidate inhibitors of serine hydrolases for which detailed functions can be largely unknown. As described therein, libraries of carbamates were evaluated versus libraries of serine hydrolases, and specific inhibitors of certain of the serine hydrolases were identified. Investigation of promising inhibitors of physiologically significant serine hydrolases has continued.
For more than 40 years, it has been known that tumor cells show dramatic elevations in their neutral ether lipid (NEL) content. Snyder and colleagues in the 1960s first reported that rodent and human tumors possess significantly higher levels of NELs relative to normal tissue (Snyder and Wood, 1969; Wood and Snyder, 1967). This finding has been confirmed for a wide range of cancer cells and primary tumors from several tissues of origin (Albert and Anderson, 1977; Lin et al., 1978; Roos and Choppin, 1984). Evidence has also emerged to suggest a pro-tumorigenic function for NELs, including a study where the levels of these lipids were found to correlate closely with tumorigenicity across a panel of mouse fibroblast cell lines (Roos and Choppin, 1984). However, the enzymes responsible for regulating NEL metabolism in cancer cells are, for the most part, poorly understood.
We have recently determined that the previously uncharacterized transmembrane enzyme KIAA1363 (also called AADACL1) controls the production of the monoalkylglycerol ether (MAGE) class of NELs in cancer cells (Chiang et al., 2006). Serine hydrolase KIAA1363 acts as a 2-acetyl MAGE hydrolase (Chiang et al., 2006) and is likely the principal source for this activity in tumor cells, which was originally detected by Snyder's group in the early 1990s (Blank et al., 1990). MAGEs can be further converted by cancer cells into the bioactive lysophospholipids alkyl-lysophosphatidyl choline (alkyl-LPC) and alkyl-lysophosphatidic acid (alkyl-LPA) (Chiang et al., 2006). Stable knockdown of KIAA1363 expression impaired tumor cell migration and tumor growth in vivo, suggesting a potentially key role for this enzyme in promoting cancer pathogenesis. It has also been found that KIAA1363 is highly elevated in aggressive breast, melanoma, ovarian (Chiang et al., 2006; Jessani et al., 2002), and pancreatic (Iacobuzio-Donahue et al., 2002) cancer cells, as well as primary breast (Ferguson et al., 2005; Jessani et al., 2005) and ovarian (Haverty et al., 2009) tumors.